The overall objective of the research proposed in this grant is to elucidate the biochemical and regulatory mechanisms of a eukaryotic transposable element in a higher metazoan using an integrated approach that combined biochemistry, genetics and molecular biology. Our efforts will be focused on understanding the biochemical mechanism of P element transposition and the mechanisms by which these eukaryotic DNA rearrangement reactions are regulated in the fruit fly, Drosophila melanogaster. DNA rearrangements play a variety of roles in the normal development and genomic flux of eukaryotic cells. These studies will also provide necessary information for the development of P elements as genetic tools in other organisms. In order to accomplish our overall objective, we will: 1.Express the Drosophila P element 87kD transposase and 66kD repressor proteins and shorter peptides in mammalian or E. coli host-vector systems. 2.Develop rapid immunoaffinity or affinity chromatography methods to facilitate the biochemical analysis of the P element proteins. 3.Analyze the biochemical activities and properties of the P element proteins as they relate to their biological role in transposition and its regulation. 4.Define the domain structures of transposase and the 66kD proteins; determine the location of the DNA and nucleotide ligand binding sites. 5.Analyze the biochemical and genetic properties of the DNA binding region(s), the nucleotide binding domain and leucine zipper regions of the P element proteins. 6.Develop an in vitro assay for P element transposition and analyze the mechanism and regulation of transposition. 7.Identify other proteins that interact with the P element proteins using genetic and biochemical methods and explore their role in transposition. 8.Develop P element-mediated transposition as a genetic tool in other organisms.